1999 Media Guide

1999 Men\u27s Track and Field Media Guide, George Fox College

Validation tumor clinical characteristics.

<p>Validation tumor clinical characteristics.</p

Expression Microarray Analysis Reveals Alternative Splicing of <i>LAMA3</i> and <i>DST</i> Genes in Head and Neck Squamous Cell Carcinoma

<div><p>Purpose</p><p>Prior studies have demonstrated tumor-specific alternative splicing events in various solid tumor types. The role of alternative splicing in the development and progression of head and neck squamous cell carcinoma (HNSCC) is unclear. Our study queried exon-level expression to implicate splice variants in HNSCC tumors.</p><p>Experimental Design</p><p>We performed a comparative genome-wide analysis of 44 HNSCC tumors and 25 uvulopalatopharyngoplasty (UPPP) tissue samples at an exon expression level. In our comparison we ranked genes based upon a novel score—the Maximum-Minimum Exon Score (MMES) – designed to predict the likelihood of an alternative splicing event occurring. We validated predicted alternative splicing events using quantitative RT-PCR on an independent cohort.</p><p>Results</p><p>After MMES scoring of 17,422 genes, the top 900 genes with the highest scores underwent additional manual inspection of expression patterns in a graphical analysis. The genes <i>LAMA3, DST, VEGFC, SDHA, RASIP1</i>, and <i>TP63</i> were selected for further validation studies because of a high frequency of alternative splicing suggested in our graphical analysis, and literature review showing their biological relevance and known splicing patterns. We confirmed <i>TP63</i> as having dominant expression of the short <i>DeltaNp63</i> isoform in HNSCC tumor samples, consistent with prior reports. Two of the six genes (<i>LAMA3</i> and <i>DST</i>) validated by quantitative RT-PCR for tumor-specific alternative splicing events (Student's t test, P<0.001).</p><p>Conclusion</p><p>Alternative splicing events of oncologically relevant proteins occur in HNSCC. The number of genes expressing tumor-specific splice variants needs further elucidation, as does the functional significance of selective isoform expression.</p></div

Demographic characteristics of discovery tumor and UPPP normal tissue samples.

<p>Demographic characteristics of discovery tumor and UPPP normal tissue samples.</p

Innovative Delivery of siRNA to Solid Tumors by Super Carbonate Apatite

<div><p>RNA interference (RNAi) technology is currently being tested in clinical trials for a limited number of diseases. However, systemic delivery of small interfering RNA (siRNA) to solid tumors has not yet been achieved in clinics. Here, we introduce an <i>in vivo</i> pH-sensitive delivery system for siRNA using super carbonate apatite (sCA) nanoparticles, which is the smallest class of nanocarrier. These carriers consist simply of inorganic ions and accumulate specifically in tumors, yet they cause no serious adverse events in mice and monkeys. Intravenously administered sCA-siRNA abundantly accumulated in the cytoplasm of tumor cells at 4 h, indicating quick achievement of endosomal escape. sCA-survivin-siRNA induced apoptosis in HT29 tumors and significantly inhibited <i>in vivo</i> tumor growth of HCT116, to a greater extent than two other <i>in vivo</i> delivery reagents. With innovative <i>in vivo</i> delivery efficiency, sCA could be a useful nanoparticle for the therapy of solid tumors.</p></div

Figure 1 demonstrates the experimental flow from tissue procurement to validation of tumor-specific alternative splicing events in our study.

<p>Figure 1 demonstrates the experimental flow from tissue procurement to validation of tumor-specific alternative splicing events in our study.</p

Validation tumor clinical characteristics.

<p>Validation tumor clinical characteristics.</p

Demographic characteristics of validation tumor and UPPP normal tissue samples.

<p>Demographic characteristics of validation tumor and UPPP normal tissue samples.</p

Anti-tumor effects and functional evidence of sCA-survivin-siRNA in HCT116 and HT29 solid tumor models.

<p>(A) For western blot analysis, HCT116 cells were seeded into 6-well plates and transfected with 100 pmol/well of either control or survivin siRNA by Lp (Lipofectamine 2000) or sCA. Actin blots served as loading controls. (B) For proliferation assays, cells were uniformly seeded into 96-well plates (1 × 10⁴ cells/well), and 5 pmol/well siRNA was used. Cell viability was examined at 48 and 72 h by WST-8 assay. Data represent mean ± SEM. *<i>P</i> = 0.0294, **<i>P</i> = 0.0304 (n = 4, Wilcoxon rank test). (C) <i>In vivo</i> tumor growth. Each vehicle, carrying 15 μg of control siRNA or survivin siRNA, was administered by intravenous injection to mice with HCT116 tumors. Data represent the mean ± SEM (n = 10 tumors, Wilcoxon rank test). (D) Immunostaining of survivin in the tumor tissues on day 19. Scale bar, 50 μm. (E) <i>In vivo</i> live imaging of sCA-siRNA (6-FAM labeled) in HT29 tumor by multiphoton microscopy at 90 min. (F) Fluorescent detection of naked-siRNA (6-FAM labeled) or sCA-siRNA (6-FAM labeled) in the HT29 tumor at 4 h. Green: 6-FAM labeled siRNA; Red: microvasculature; Blue: DAPI stained nuclei. Scale bar, 50 μm. (G) Mice were administered with 40 μg of naked-survivin-siRNA or sCA-survivin-siRNA on days 0, 1, and 2. Tumors were removed on day 3, and western blot analysis for survivin was performed. (H) Many tumor cells treated with sCA-survivin-siRNA (6-FAM labeled) had condensed nuclei and were positive for TUNEL assay. Scale bar, 50 μm.</p

<i>In vitro</i> transfection efficiency by lipofectamine 2000 (Lp) or sCA.

<p>(A) HCT116 was examined for transfection efficiency of 6-FAM labeled siRNA (20 pmol/well) by Lp or sCA. Cells were analyzed with flow cytometry at 4 and 12 h and then further observed by fluorescence microscopy. Bright field images are shown in the right panels. MFI; mean fluorescence intensity. Scale bars, 300 μm. (B) The relative MFI of Lp-siRNA or sCA-siRNA treated cells. Data represent mean ± SEM. *<i>P</i> = 0.0304, **<i>P</i> = 0.0294 (n = 4, Wilcoxon rank test). (C) Uptake behavior of Lp-siRNA or sCA-siRNA in HCT116 cells analyzed by confocal laser scanning microscopy at 4 and 12 h. Merged panels show fluorescent siRNA (green) and DAPI nuclear staining (blue). Scale bars, 10 μm.</p